Preventing conversion of citrulline to argininosuccinate to limit pathological nitric oxide overproduction

ABSTRACT

Administration of argininosuccinate synthetase activity reducing agents, e.g., argininosuccinate synthetase induction blocking agents (e.g., antibiotics that bind to DNA sequences present in the upstream regulatory region of the argininosuccinate synthetase gene, such as mithramycin) and argininosuccinate synthetase inhibitors (e.g., L-citrulline antagonists such as methyl citrulline and L-aspartate antagonists such as D-aspartate) is useful to prevent or treat sepsis or cytokine-induced systemic hypotension, is useful in the treatment of sepsis or cytokine-induced systemic hypotension to restore vascular sensitivity to the effects of α 1  -adrenergic agonists, and is useful to suppress an immune response, e.g., in treating inflammation. In one embodiment, certain argininosuccinate synthetase activity reducing agents are used together with arginine antagonists to treat sepsis or cytokine induced hypotension.

This invention was made at least in part with Government support underNational Institutes of Health Grant HL46403 and under NationalInstitutes of Health Grant DK37116.

TECHNICAL FIELD

This invention is directed to a novel approach for inhibiting biologicalnitric oxide production.

BACKGROUND OF THE INVENTION

For several decades nitroglycerin has been administered to humans as avasodilating agent in the treatment of cardiovascular disease. It hasbeen shown that nitroglycerin so administered is converted in the bodyto nitric oxide which is the pharmacologically active metabolite.Recently, nitric oxide has been shown to be formed enzymatically as anormal metabolite from arginine in vascular endothelium to provide animportant component of endothelium-derived relaxing factors (EDRFs)which are currently being intensively studied as participating inregulation of blood flow and vascular resistance. Macrophages have alsobeen shown to produce nitric oxide in the body as a component of theircell killing and/or cytostatic function.

More recently it has been established that the enzyme forming nitricoxide from arginine, i.e., nitric oxide synthase, occurs in at least twodistinct types, namely the constitutive forms and an inducible form.Constitutive forms are present in normal endothelial cells, neurons andsome other tissues. Formation of nitric oxide by a constitutive form inendothelial cells is thought to play a role in normal blood pressureregulation. The inducible form of nitric oxide synthase has been foundto be present in activated macrophages and is induced in endothelialcells and vascular smooth muscle cells, for example, by variouscytokines and/or microbial products. It is thought that in sepsis orcytokine-induced shock, overproduction of nitric oxide by the inducibleform of nitric oxide synthase plays an important role in the observedlife-threatening hypotension. Furthermore, it is thought thatoverproduction of nitric oxide by the inducible form of nitric oxidesynthase is a basis for insensitivity to clinically used pressor agentssuch as α₁ -adrenergic agonists in the treatment of septic orcytokine-induced shock patients. Moreover, it is thought thatoverproduction of nitric oxide by inducible form of nitric oxidesynthase is involved in inflammation incident to an immune response.

Overproduction of nitric oxide is due either to overstimulation of aconstitutive nitric oxide synthase (cNOS) or to overexpression ofinducible nitric oxide synthase (iNOS). In either case, overproductionof nitric oxide is also dependent on adequate availability of arginine,the substrate of nitric oxide synthase. Arginine supply is maintained inthree ways: (i) protein degradation, (ii) uptake of arginine fromplasma, (iii) conversion of citrulline to arginine by pathways involvingconversion of citrulline and aspartate to argininosuccinate (ASA) byargininosuccinate synthetase and conversion of ASA to arginine andfumarate by argininosuccinate lyase. In the case of iNOS, enzymeactivity and overproduction of nitric oxide is also dependent onavailability of required cofactors including tetrahydrobiopterin.

To date, pathological overproduction of nitric oxide has been controlledby administration of nitric oxide synthase inhibiting arginineantagonists, plasma arginine depleting enzymes and tetrahydrobiopterininduction or utilization blocking agents.

SUMMARY OF THE INVENTION

It has been discovered herein that administration of argininosuccinatesynthetase activity reducing agents is useful to prevent or treat sepsisor cytokine-induced systemic hypotension, is useful in the treatment ofsepsis or cytokine-induced systemic hypotension to restore vascularsensitivity to the effects of α₁ -adrenergic agonists, is useful tosuppress an immune response, e.g., in treating inflammation, and isuseful to prevent or treat a subject for a stroke.

One embodiment herein is directed to a method of prophylaxis ortreatment of a subject for systemic hypotension caused by pathologicaloverproduction of nitric oxide from arginine in vascular cells in saidsubject induced by therapy with a cytokine or by exposure to a bacterialendotoxin, said method comprising administering to a subject expected todevelop or having such systemic hypotension a therapeutically effectiveamount of an argininosuccinate synthetase activity reducing agent. Theagent can be either an argininosuccinate synthetase induction blockingagent or an argininosuccinate synthetase inhibitor. A therapeuticallyeffective amount of an argininosuccinate synthetase activity reducingagent in this method is one that causes reduction in induced nitricoxide production to the extent of causing an increase in blood pressure.

Another embodiment herein is directed to a method for treatment of asubject for systemic hypotension caused by pathological overproductionof nitric oxide from arginine in vascular cells in said subject inducedby therapy with a cytokine or by exposure to a bacterial endotoxin, saidmethod comprising administering to a subject having such systemichypotension a therapeutically effective amount of an α₁ -adrenergicagonist, and an amount of argininosuccinate synthetase activity reducingagent to restore vascular contractile sensitivity to the effects of saidα₁ -adrenergic agonist. The argininosuccinate synthetase activityreducing agent can be either an argininosuccinate synthetase inductionblocking agent or an argininosuccinate synthetase inhibitor. Atherapeutically effective amount of α₁ -adrenergic agonist is one thatcauses increase in blood pressure.

Still another embodiment herein is directed to a method for treating asubject for systemic hypotension caused by pathological overproductionof nitric oxide from arginine in vascular cells in said subject inducedby therapy with a cytokine or by exposure to a bacterial endotoxin, saidmethod comprising administering to said subject a therapeuticallyeffective amount of an arginine antagonist of nitric oxide synthesis bynitric oxide synthase and a therapeutically effective amount of anargininosuccinate synthetase activity reducing agent selected from thegroup consisting of argininosuccinate synthetase induction blockingagents and argininosuccinate synthetase inhibitors which do not blockthe activity of inducible nitric oxide synthase (by inhibiting induciblenitric oxide synthase by binding to it or its heme cofactor). Thetherapeutically effective amount of arginine antagonist of nitric oxidesynthesis by inducible nitric oxide synthase is an induced nitric oxideproduction limiting amount, i.e., a blood pressure raising amount. Thetherapeutically effective amount of the argininosuccinate synthetaseactivity reducing agent in this method is that which when administeredto a subject sufficiently limits the induction, or further induction, ofargininosuccinate synthetase or inhibits the activity ofargininosuccinate synthetase such that the induced argininosuccinatesynthetase dependent citrulline to arginine metabolic pathway does notcontribute significantly to the availability of arginine needed tosustain overproduction of nitric oxide by inducible nitric oxidesynthase, thereby to potentiate duration or magnitude of the bloodpressure increase caused by the administration of the arginineantagonist of nitric oxide synthesis by nitric oxide synthase.

Therapy with a cytokine includes therapy with interferons includinginterferon-gamma, tumor necrosis factor, interleukin-1 or interleukin-2,e.g., chemotherapeutic treatment with tumor necrosis factor orinterleukin-2. Treatment with nitric oxide synthase inducing cytokineswill cause systemic hypotension by pathological overproduction of nitricoxide if continued for a long enough period. Thus, the expectation ofsystemic hypotension caused by pathological overproduction of nitricoxide is currently a limitation on the extent and duration of cytokinetherapy.

Bacterial endotoxin induced system hypotension, also known as sepsis orseptic shock, may arise, e.g., from bacterial infection orimmunosuppression therapy. There are subjects that are expected todevelop this condition, i.e., who are at risk for developing thiscondition, e.g., those suffering from burn or hemorrhage or undergoingimmunosuppression therapy.

In another embodiment, the invention herein is directed to a method ofprophylaxis or treatment of a subject for inflammation caused by inducednitric oxide production from arginine in cells, said method comprisingadministering to a subject at risk for or having such inflammation, atherapeutically effective amount of an argininosuccinate synthetaseactivity reducing agent. The inflammation may arise, e.g., fromnon-acute allergic reactions including contact dermatitis, fromautoimmune conditions including rheumatoid arthritis, myasthenia gravis,multiple schlerosis, lupus erythematosus and Parkinson's disease, andhost-defense immune mechanisms, e.g., allograft rejection reactions,mediated by immunologically induced nitric oxide production. Those atrisk for such condition, e.g., those of high probability to becomeafflicted with this condition include those undergoing allografttreatment. The agent can be either an argininosuccinate synthetaseinduction blocking agent or an argininosuccinate synthetase inhibitor. Atherapeutically effective amount of an argininosuccinate synthetaseactivity reducing agent in this method is an amount which causesreduction in induced nitric oxide synthesis to the extent of suppressingthe immune response that is part of the inflammatory response therebyattenuating the inflammation.

In another embodiment, the invention herein is directed to a method ofprophylaxis or treatment of a subject for a stroke, said methodcomprising administering to said subject a therapeutically effectiveamount, e.g., a neuronal cell protecting amount, of an argininosuccinatesynthetase activity reducing agent.

In another embodiment, the invention is directed at N.sup.ω -C₁₋₆-alkyl-L-citrullines as novel compounds.

The term "subject" is used herein to mean a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of concentration of mithramycin versusargininosuccinate synthetase activity (filled in circles) and versusnitric oxide synthase activity (open circles) and depicts results ofExample I.

FIG. 2 shows the time course oflipopolysaccharide/interferon-gamma-induced changes in mRNA levels forargininosuccinate synthetase (AS), argininosuccinate lyase (AL), nitricoxide synthase (NOS) and glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and depicts results of Example II.

FIG. 3 effect of the change concentration of mithramycin on levels ofargininosuccinate synthetase mRNA and nitric oxide synthase mRNA and onGTP cyclohydrolase I mRNA in induced rat aortic smooth muscle cells asdetermined by reverse transcriptase-polymerase chain reaction analysisand depicts results of Example III.

FIG. 4 depicts graphs of mithramycin concentration versus nitriteproduction with mithramycin administered post LPS/interferon gammaadministration (open circles) and administered concurrently withLPS/interferon gamma (closed circles), and depicts results of ExampleIV.

FIG. 5 depicts graphs showing the effect on nitrite production ininduced intact cells of varying the concentration of citrulline at fixedconcentrations of arginine and depicts results of Example V.

DETAILED DESCRIPTION

The argininosuccinate synthetase activity reducing agents herein consistof argininosuccinate synthetase induction blocking agents andargininosuccinate synthetase inhibitors. They do not include agentswhich globally inhibit gene transcription such as actinomycin D, oragents which globally inhibit protein synthesis such as cycloheximide,or glucocorticoids.

The argininosuccinate synthetase induction blocking agents arepreferably antibiotics that bind to DNA sequences present in theupstream regulatory region of the argininosuccinate synthetase gene(described in Freytag, S. O., et al, J. Biol. Chem., 259(5), 3160-3166,3/84 and Jinno, Y., et al, J. Biochem. 98, 1395-1403, 1985 and isarchived in The European Molecular Biology Database under EMBL accessionnumber 03258), such as the sequences for the binding of thetranscriptional activating factor SP1. Antibiotics known to do thisinclude mithramycin (also known as plicamycin), chromomycins (e.g.,chromomycin A₃) and olivomycins (e.g., olivomycins A, B, C and D). Someof these antibiotics also have been found to block the induction ofnitric oxide synthase gene expression. Such antibiotics limit inducedproduction of nitric oxide in two ways, namely by limiting induction ofargininosuccinate synthetase resulting in limiting of production ofsubstrate arginine from citrulline and also by limiting induction ofnitric oxide synthase catalyst so that the conversion of arginine intonitric oxide and citrulline is limited. Administration of theseantibiotics also has the effect of depleting calcium levels. However,this is not a problem when these antibiotics are administered to treatacute conditions (e.g., when used alone or together with α₁ -adrenergicagonists in treatment of sepsis or cytokine-induced hypotension) or forshort term treatment for prophylaxis of expected induced hypotension orfor short term treatment of inflammation.

The argininosuccinate synthetase inhibitors bind to argininosuccinatesynthetase to block the activity thereof and thereby prevent theargininosuccinate synthetase from converting citrulline toargininosuccinate.

The argininosuccinate synthetase inhibitors can be, for example,L-citrulline antagonists, i.e., compounds which interfere with theaction of argininosuccinate synthetase on L-citrulline, or L-aspartateantagonists, i.e., compounds which interfere with the action ofargininosuccinate synthetase on L-aspartate.

The L-citrulline antagonists include, for example, L-thiocitrulline andL-homothiocitrulline which also block the activity of inducible nitricoxide synthase by binding to heme cofactor of inducible nitric oxidesynthase (i.e., the catalytically important heme cofactor enfolded in anitric oxide synthase molecule) and canavanine which also inhibitsinducible nitric oxide synthase as well as L-citrulline antagonistsdifferent from L-thiocitrulline and L-homothiocitrulline and canavanineand which do not also block the activity of nitric oxide synthase bybinding to inducible nitric oxide synthase or its heme cofactor, e.g.,N.sup.ω -C₁₋₆ -alkyl-L-citrullines, such as N.sup.ω-methyl-L-citrulline, and amino acids which naturally occur in mammalianspecies, except for arginine, preferably L-norvaline, L-isoleucine,L-valine and L-serine.

The L-aspartate antagonists include, for example,α-methyl-D-L-aspartate, D-aspartate, erythro-β-hydroxyl-DL-aspartate,threo-β-hydroxy-β-methyl-DL-aspartate anderythro-β-hydroxy-β-methyl-DL-aspartate.

For the embodiment where α₁ -adrenergic agonists are utilized, α₁-adrenergic agonists are used for the same purpose now (i.e., toincrease blood pressure in a hypotensive subject) but eventually stopworking because of loss of vascular contractile sensitivity. The α₁-adrenergic agonists include epinephrine, norepinephrine, dopamine,phenylephrine, metaraminol, methoxamine, ephedrine and nephentermine.

For the embodiment where arginine antagonists of nitric oxide synthesisby nitric oxide synthase are used, i.e., compounds which interfere withthe action of iNOS on L-arginine, these bind to iNOS to block theactivity thereof and thereby prevent the iNOS from converting arginineto nitric oxide and citrulline. The arginine antagonists contemplatedare those which are not also argininosuccinate synthetase inhibitors.These include N^(G) -methyl-L-arginine, N^(G) -amino-L-arginine, N^(G)-nitro-L-arginine, N^(G) -nitro-L-arginine methyl ester, N.sup.δ-iminomethyl-L-ornithine, N⁶ -(hydrazinoiminomethyl)lysine, andguanidino substituted arginines or homoarginines based on monoalkylcarbon-substituted ornithines or lysines (as described in U.S. Pat. No.5,281,627).

We turn now to the method herein for the prophylaxis or treatment of asubject for systemic hypotension caused by pathological overproductionof nitric oxide from arginine in vascular cells in said subject inducedby therapy with a cytokine or by exposure to a bacterial endotoxin, saidmethod comprising administering to a subject expected to develop orhaving such systemic hypotension a therapeutically effective amount ofargininosuccinate synthetase activity reducing agent. We turn firstly towhere argininosuccinate synthetase induction blocking agents are used asthe argininosuccinate synthetase activity reducing agents. Mithramycinis utilized preferably at a dosage ranging from 15 to 25 μg/kg for acutetreatments (e.g., treatment of hypotension resulting from septic shock)and 12 to 15 μg/kg for prophylaxis and may be administered intravenouslyas a bolus injection or as an infusion over 1 to 3 hours. Chromomycinsare utilized preferably at a dosage ranging from about 15 to 26 μg/kgfor acute treatments and 12 to 16 μg/kg for prophylactic treatments andmay be administered intravenously. Olivomycins are utilized at a dosagepreferably ranging from 15 to 200 μg/kg for acute treatments and 12 to125 μg/kg for prophylaxis and may be administered intravenously. Asthese agents are known to lower plasma levels of calcium, calciumsupplementation may be provided to patients in need thereof. Forprophylaxis, the argininosuccinate synthetase induction blocking agentsshould be administered at the dosages recited for this purpose daily forno more than about 4 days. We turn now to where argininosuccinatesynthetase inhibitors are used as the argininosuccinate synthetaseactivity reducing agents. Where L-citrulline antagonists are used as theargininosuccinate synthetase inhibitors, for treatment of systemichypotension already occurring, these are administered in a bloodpressure raising amount, generally 1 mg/kg to 100 mg/kg for L-enantiomer(preferably 2 mg/kg to 20 mg/kg for L-thiocitrulline and 2 mg/kg to 40mg/kg for L-homothiocitrulline and 20 mg/kg to 100 mg/kg for N.sup.ω-methyl-L-citrulline) by a route of administration obtaining a fastresponse, normally parenteral, preferably intravenous, and for treatmentin cases where systemic hypotension is expected (i.e., for prophylaxis,i.e., prevention or delay of the condition occurring or to providereduced severity of the condition which occurs), these are administeredto provide a plasma level of L-citrulline antagonist sufficient toeliminate or delay the occurring of the hypotension or reduce theseverity of the hypotension which occurs, generally a plasmaconcentration of L-citrulline antagonist ranging from 1 μM to 1000 μMfor L-enantiomer (preferably 10 μM to 50 μM for L-thiocitrulline andL-homothiocitrulline and 200 to 1000 μM for N.sup.ω-methyl-L-citrulline) by a route of administration which can beparenteral (e.g., intravenous) but also can be oral (doses to providethis concentration may be determined by considering the half-life of thecompounds in the body). L-Citrulline antagonists can be administered forthe same purposes by continuous infusion, in which case the doses wouldrange from 1 mg/kg/hr to 100 mg/kg/hr generally, (preferably 2 mg/kg/hrto 20 mg/kg/hr for L-thiocitrulline and 2 mg/kg/hr to 40 mg/kg/hr forL-homothiocitrulline and 20 mg/kg/hr to 100 mg/kg/hr for N.sup.ω-methyl-L-citrulline). Where L-aspartate antagonists are used as theargininosuccinate synthetase inhibitors, for treatment of systemichypotension already occurring, these are administered in a bloodpressure raising amount, generally 10 mg/kg to 1000 mg/kg (preferably 10mg/kg to 100 mg/kg for D-aspartate) by a route of administrationobtaining a fast response, normally parenteral, preferably intravenous,and for treatment in cases where systemic hypotension is expected (i.e.,for prophylaxis), these are administered to provide a plasma level ofL-aspartate antagonist sufficient to eliminate or delay the occurring ofthe hypotension or reduce the severity of the hypotension which occurs,generally a plasma concentration of L-aspartate antagonist ranging from50 μM to 1000 μM (preferably 50 μM to 200 μM for D-aspartate) by a routeof administration which can be parenteral (e.g., intravenous) but alsocan be oral (doses to provide the concentration may be determined byconsidering the half-life of the compounds in the body). L-Aspartateantagonists can be administered for the same purposes by continuousinfusion, in which case the doses would range from 10 mg/kg/hr to 1000mg/kg/hr (preferably 10 mg/kg/hr to 100 mg/kg/hr for D-aspartate).

We turn now to the method herein for the treatment of a subject forsystemic hypotension caused by pathological overproduction of nitricoxide from arginine in vascular cells in said subject induced by therapywith a cytokine or by exposure to a bacterial endotoxin, said methodcomprising administering to a subject having such systemic hypotension atherapeutically effective amount of an α₁ -adrenergic agonist and anamount of argininosuccinate synthetase activity reducing agent torestore vascular contractile sensitivity to the effects of the α₁-adrenergic agonist, hereinafter referred to as the second methodherein.

The α₁ -adrenergic agonists for the second method herein are used in thesame dosages as they are now used for the same purpose (i.e., toincrease blood pressure in a hypotensive patient), i.e., in conventionaltherapeutically effective amounts. Doses for the α₁ -adrenergic agonistdopamine typically range from 2 μg/kg/min to 50 μg/kg/min. Doses for theα₁ -adrenergic agonist norepinephrine typically range from 2 μg/min to 4μg/min. Doses for the α₁ -adrenergic agonist phenylephrine can rangefrom 0.1 to 10 μg/kg. The route of administration of the most popular α₁-adrenergic agonists (norepinephrine and dopamine) is intravenous andfor the others the route of administration is intravenous or in somecases subcutaneous.

We turn now to where argininosuccinate synthetase induction blockingagents are used as the argininosuccinate synthetase activity reducingagents in the second method herein. Mithramycin is utilized at a dosagepreferably ranging from 15 to 25 μg/kg and may be administeredintravenously as a bolus injection or as an infusion over 1 to 3 hours.Chromomycins are utilized preferably at a dosage ranging from about 15to 26 μg/kg and may be administered intravenously. Olivomycins areutilized at a dosage preferably ranging from 15 to 200 μg/kg and may beadministered intravenously.

We turn now to where argininosuccinate synthetase inhibitors are used asthe argininosuccinate synthetase activity reducing agents in the secondmethod herein. Where L-citrulline antagonists are used as theargininosuccinate synthetase inhibitors, these are administered in avascular contractile sensitivity restoring amount, generally 1 mg/kg to100 mg/kg for L-enantiomer (preferably 2 mg/kg to 20 mg/kg forL-thiocitrulline and L-homothiocitrulline and 20 mg/kg to 100 mg/kg forN.sup.ω -methyl-L-citrulline) by a route of administration obtaining afast response, normally parenteral, preferably intravenous. L-Citrullineantagonists can be administered for the same purpose by continuousinfusion, in which case the doses would range from 1 mg/kg/hr to 100mg/kg/hr generally (preferably 2 mg/kg/hr to 20 mg/kg/hr forL-thiocitrulline and L-homothiocitrulline and 20 mg/kg/hr to 100mg/kg/hr for N.sup.ω -methyl-L-citrulline). Where L-aspartateantagonists are used as the argininosuccinate synthetase inhibitors,these are administered in a vascular contractile sensitivity restoringamount, generally 10 mg/kg to 1000 mg/kg (preferably 10 mg/kg to 100mg/kg for D-aspartate) by a route of administration obtaining a fastresponse, normally parenteral, preferably intravenous. L-Aspartateantagonists can be administered for the same purposes by continuousinfusion in which case the doses would range from 10 mg/kg/hr to 1000mg/kg/hr (preferably 10 mg/kg/hr to 100 mg/kg/hr for D-aspartate).

We turn now to the method herein for the treatment of a subject forsystemic hypotension caused by pathological overproduction of nitricoxide from arginine in vascular cells in said subject induced by therapywith a cytokine or by exposure to a bacterial endotoxin, said methodcomprising administering to said subject a therapeutically effectiveamount of an arginine antagonist of nitric oxide synthesis by nitricoxide synthase and a therapeutically effective amount of anargininosuccinate synthetase activity reducing agent selected from thegroup consisting of argininosuccinate synthetase induction blockingagents and argininosuccinate synthetase inhibitors which do not blockthe activity of inducible nitric oxide synthase (by inhibiting induciblenitric oxide synthase by binding to it or its heme cofactor),hereinafter referred to as the third method herein.

The arginine antagonist competes with arginine for the active site ofnitric oxide synthase. Therefore, lowering intracellular arginine levelby administering an argininosuccinate synthetase activity reducing agentenables lowering the dosage of arginine antagonist necessary to limitinduced nitric oxide production and raise blood pressure, e.g., to lessthan 50% of the dosage of arginine antagonist otherwise necessary. Thisdiminishes the potential toxicity of the arginine antagonist.

The arginine antagonists are used in the third method herein in a bloodpressure raising amount. The dosages generally range from 0.1 to 100mg/kg and/or from 0.1 to 100 mg/kg/hr, with the actual dosage dependingon the arginine antagonist selected. Doses for N^(G) -methyl-L-argininerange from 1 to 40 mg/kg and/or 1 to 40 mg/kg/hr. The route ofadministration is preferably intravenous or other route providing a fastresponse.

The argininosuccinate synthetase induction blocking agents for the thirdmethod herein are those generally described above. The argininosuccinatesynthetase inhibitors which do not block the activity of induciblenitric oxide synthase (by inhibiting inducible nitric oxide synthase bybinding to it or its heme cofactor) for the third method herein can be(1) L-citrulline antagonists which do not block the activity of iNOS bybinding to iNOS or its heme cofactor, or (2) L-aspartate antagonists.The L-citrulline antagonists for the third method herein can be, forexample, N¹⁰⁷ -C₁₋₅ -alkyl-L-citrullines such as N.sup.ω-methyl-L-citrulline and amino acids which naturally occur inmammalian-species, except for arginine, preferably L-norvaline,L-isoleucine, L-valine and L-serine. L-Aspartate antagonists aregenerally described above.

Argininosuccinate synthetase activity reducing agents are used in thethird method herein in an amount such that the induced argininosuccinatesynthetase dependent citrulline to arginine metabolic pathway does notcontribute significantly to the availability of arginine needed tosustain overproduction of nitric oxide by nitric oxide synthase, as maybe determined by the ability to administer less arginine antagonist toachieve the same result as administering more arginine antagonistwithout the argininosuccinate synthetase activity reducing agent beingadministered.

We turn now to when argininosuccinate synthetase induction blockingagents are used as the argininosuccinate synthetase activity reducingagents in the third method herein. Mithramycin is utilized preferably ata dosage ranging from about 12 to 15 μg/kg and may be administeredintravenously as a bolus injection or as an infusion over 1 to 3 hours.Chromomycins are utilized preferably at a dosage ranging from about 15to 26 μg/kg and may be administered intravenously. Olivomycins areutilized preferably at a dosage ranging from about 12 to 125 μg/kg andmay be administered intravenously.

We turn now to where argininosuccinate synthetase inhibitors which donot block the activity of inducible nitric oxide synthase are used inthe third method herein. The L-citrulline antagonists are generallyadministered in a dosage of 1 mg/kg to 100 mg/kg (preferably 20 mg/kg to100 mg/kg for N.sup.ω -methyl-L-citrulline) by a route of administrationobtaining a fast response, normally parenteral, preferably intravenous.The L-citrulline antagonists can be administered for the same purpose bycontinuous infusion, in which case the doses would range from 1 mg/kg/hrto 100 mg/kg/hr generally (preferably, 20 mg/kg/hr to 100 mg/kg/hr forN.sup.ω -methyl-L-citrulline. The L-aspartate antagonists are generallyadministered in a dosage of 10 mg/kg to 1000 mg/kg (preferably 10 mg/kgto 100 mg/kg for D-aspartate) by a route of administration obtaining afast response, normally parenteral, preferably intravenous. TheL-aspartate antagonists can be administered for the same purposes bycontinuous infusion, in which case the doses would range from 10mg/kg/hr to 1000 mg/kg/hr (preferably 10 mg/kg/hr to 100 mg/kg/hr forD-aspartate.

We turn now to the method herein of prophylaxis or treatment of asubject for inflammation caused by induced nitric oxide production fromarginine in immune cells, said method comprising administering to asubject at risk for or having such inflammation, a therapeuticallyeffective amount of an argininosuccinate synthetase activity reducingagent, hereinafter referred to as the fourth method herein.

We turn now to where argininosuccinate synthetase induction blockingagents are utilized as the argininosuccinate synthetase activityreducing agents in the fourth method herein. Mithramycin is utilized ata dosage ranging from about 0.1 to 50 μg/kg, for intravenousadministration preferably at a dosage ranging from about 12 to 15 μg/kg.Chromomycins are utilized at a dosage ranging from about 0.1 to 50μg/kg, for intravenous administration preferably at a dosage rangingfrom about 12 to 16 μg/kg. Olivomycins are utilized at a dosage rangingfrom about 0.1 to 125 μg/kg, for intravenous administration preferablyranging from about 12 to 125 μg/kg. Methods of administration includeoral, intramuscular, subcutaneous, intrasynovial and intravenous. Thedosages set forth are daily dosages and are administered for prophylaxispurposes or for a period of time to cause suppression of immune responseand attenuation of inflammation, normally 2 days or more but for no morethan 7 days.

We turn now to where argininosuccinate synthetase inhibitors areutilized as the argininosuccinate synthetase activity reducing agents inthe fourth method herein. Where L-citrulline antagonists are used as theargininosuccinate synthetase inhibitors, for treatment of inflammation,these are administered in an inflammation attenuating amount, generally0.01 mg/kg to 100 mg/kg and/or 0.01 mg/kg/hr to 100 mg/kg/hr forL-enantiomer (preferably 0.1 mg/kg to 20 mg/kg and/or 0.1 mg/kg/hr to 20mg/kg/hr for L-thiocitrulline and L-homothiocitrulline and 0.2 mg/kg to100 mg/kg and/or 0.2 mg/kg/hr to 100 mg/kg/hr for N.sup.ω-methyl-L-citrulline). Where L-citrulline antagonists are used as theargininosuccinate synthetase inhibitors, for prophylaxis of inflammationfrom induced production of nitric oxide, these are administered toprovide a plasma level of L-citrulline antagonist sufficient toeliminate or delay the occurring of inflammation from induced nitricoxide production or reduce the severity of the inflammation whichoccurs, generally a plasma concentration of L-citrulline antagonistranging from 1 μM to 1000 μM for L-enantiomer (preferably 10 μM to 50 μMfor L-thiocitrulline and L-homothiocitrulline and 200 to 1000 μM forN.sup.ω -methyl-L-citrulline). Where L-aspartate antagonists are used asthe argininosuccinate synthetase inhibitors, these are administered inan inflammation attenuating amount, generally 0.1 mg/kg to 1000 mg/kgand/or 0.1 mg/kg/hr to 1000 mg/kg/hr (preferably 0.1 mg/kg to 100 mg/kgand/or 0.1 mg/kg/hr to 100 mg/kg/hr for D-aspartate). Where L-aspartateantagonists are used as the argininosuccinate synthetase inhibitors, forprophylaxis of inflammation from induced production of nitric oxide,these are administered to provide a plasma level of L-aspartateantagonist sufficient to eliminate or delay the occurring ofinflammation from induced nitric oxide production or reduce the severityof the inflammation which occurs, generally a plasma concentration ofL-aspartate antagonist ranging from 50 to 1000 μM (preferably 50 to 200μM for D-aspartate). Methods of administration include oral,intramuscular, subcutaneous, intrasynovial and intravenous. The dosagesrecited, which may be repeated, are administered for a period of time tocause suppression of immune response and attenuation of inflammation,i.e., treating for two days or more, e.g., for two to three weeks. Forprophylaxis, doses to provide the aforestated plasma concentrations maybe determined by considering the half-life of the compounds in the body.

The invention is illustrated in the following examples.

EXAMPLE I

Aortic smooth muscle cells were cultured by explanting segments of themedial layer of aortae from adult male Fischer 344 rats. Aortae wereremoved aseptically and freed of adventitial and endothelial cells byscraping both the lumenal and abluminal surfaces. Medial fragments (1-2mm) were allowed to attach to dry Primaria 25 cm² tissue culture flasks(Falcon; Oxnard, Calif.) which were kept moist with growth medium untilcells emerged. Cultures were fed twice weekly with medium 199 containing10% fetal bovine serum, 25 mM HEPES, 2 mM L-glutamine, 40 μg/mlendothelial cell growth supplement (Biomedical Technologies; Stoughton,Mass.) and 10 μg/ml gentamycin (GIBCO; Grand Island, N.Y.). When primarycultures became confluent, they were passaged by trypsinization and theexplants were discarded. Cells in passage 10-15 were seeded at20,000/well in 96-well plates.

When the cells became confluent (density of 60-80×10³ cells in a well),the medium was removed by suction and fresh medium consisting of 200 μlof RPMI 1640 (Whittaker Laboratories) containing 10% bovine calf serum,2.5 mM glutamine and penicillin (80 U/ml), streptomycin (80 μg/ml) andfugizone (2 μg/ml) was added to each well via a pipette.

Groups of 4 wells were administered fixed concentrations of mithramycin(0.001 to 10 μg/ml); control wells received no mithramycin. To each wasalso added bacterial lipopolysaccharide (endotoxin); Serotype: E. Coli.0111:B4, 30 μg/ml) plus rat interferon-gamma (50 ng/ml). The wells werethen incubated at 37° C. in a humidified incubator for 24 hours. Cellswere then harvested and prepared for assay of argininosuccinatesynthetase (AS activity) and nitric oxide synthase (NOS activity).

Nitric oxide formation by smooth muscle cell cytosol was measured usinga spectrophotometric kinetic microplate assay based on the increase inA₄₀₅ accompanying the oxidation of Fe²⁺ -myoglobin by nitric oxide.Cytosol (50-100 μg of protein) was incubated with 40 μmdithionite-reduced myoglobin (prepared as described in Gross, S. S., etal, Biochem. Biophys. Res. Commun. 160,881-886, 1989) in assay buffercontaining 80 mM Tris, 0.5 mM L-arginine, 0.5 mM NADPH, and 10 μmtetrahydrobiopterin (pH 7.6). Assays were performed in triplicate withA₄₀₅ recorded for each sample every 18 seconds for a period of 15minutes. The rate of nitric oxide synthesis was calculated from the rateof increase in A₄₀₅ by linear regression analysis. The conversion factorrelating A₄₀₅ to the nitric oxide synthesis rate under the conditionsemployed was 1.05 milli optical density units (also referred to as mOD)min⁻¹ pmol⁻¹ of nitric oxide formed.

Argininosuccinate synthetase activity was assayed based on theconversion of [¹⁴ C]aspartate to [¹⁴ C]argininosuccinate, as describedin O'Brien, W. E., Biochemistry 18, 5353-5356, 1979, with the exceptionthat aspartate was present at a concentration of 30 μm (0.021 μCi/nmol).Reaction mixtures also contained L-citrulline (5 mM), Tris (10 mM, pH7.5), ATP 0.1mM), phosphoenolpyruvate (1.5 mM), pyruvate kinase (4.5units), myokinase (4 units), and pyrophosphatase (0.2 unit) in a totalvolume of 0.3 ml. Reactions were allowed to proceed for 90 minutes at37° C before termination and quantification of the [¹⁴C]argininosuccinate formed, using ion exchange chromatography on Dowex1-X8 (200-400 mesh, Bio-Rad).

The results are shown in FIG. 1 where the filled in circles denoteargininosuccinate synthetase (denoted AS in FIG. 1) activity and theopen circles denote nitric oxide synthase (denoted NOS in FIG. 1)activity.

FIG. 1 shows that mithramycin, as a function of its concentration blocksargininosuccinate synthetase activity as well as nitric oxide synthaseactivity. The concentrations of mithramycin that are effective in thisregard are the same concentrations obtained in patients administereddoses of mithramycin for hypercalcemia as described in The Merck Manual,16th edition, page 1013.

Similar results of blocking argininosuccinate synthetase activity as afunction of concentration are obtained using the same concentrations ofchromomycins and with up to 5 times greater concentrations ofolivomycins.

EXAMPLE II

Rat aortic smooth muscle cells were grown as described in Example I andwere treated with 30 μg/ml bacterial lipopolysaccharide in combinationwith 50 ng/ml interferon-gamma. At the indicated times as shown in FIG.2, cells were harvested and RNA was prepared and assayed by reversetranscriptase-polymerase chain reaction using gene specific primers. RNAwas extracted by a modified guanidinium isothiocyanate method (RNAzol;Cinna/Biotecz, Houston, Tex.). Reverse transcriptase-polymerase chainreaction was performed by standard methods using 1 μg of total RNA. Thiswas followed by polymerase chain reaction amplification using syntheticgene-specific primers for rat argininosuccinate synthetase as describedin Amaya, Y., et al, J. Biochem. (Tokyo) 103, 177-181 (1988), forargininosuccinate lyase as described in Surh, L. C., et al, NucleicAcids Research, 16, 9352 (1988) and murine-inducible nitric oxidesynthase as described in Lyons, C. R., et al, J. Biol. Chem. 267,6370-6374 (1992). The primers are specifically described in Hattori, Y.,et al, J. Biol. Chem. 269, 9405-9408 (1994). Polymerase chain reactionamplification was performed according to the following schedule:denaturation, annealing, and elongation at 95, 55 and 72° C. for 30 sec,30 sec and 1 min, respectively, for 30 cycles. Parallel amplification ofglyceraldehyde-3-phosphate dehydrogenase was performed for referenceusing primers as described in Terada, Y., et al, J. Clin. Invest. 90,659-665 (1992). Polymerase chain reaction products were electrophoresedon a 1.5% agarose gel containing ethidium bromide and visualized byUV-induced fluorescence. All polymerase chain reactions resulted in theamplification of a single product of the predicted size forargininosuccinate synthetase (612 bp), argininosuccinate lyase (600 bp)and nitric oxide synthase (807 bp). To confirm the identity of thepolymerase chain reaction products, the products were cut with EcoRI andfound to each give two fragments of the expected sizes based on reportedsequences for rat argininosuccinate synthetase as described in Amaya,Y., et al, J. Biochem. (Tokyo) 103, 177-181 (1988) and for ratargininosuccinate lyase as described in Surh, L. C., et al, NucleicAcids Res. 16, 9352 (1988). Subcloning and dideoxynucleotide sequencingof the polymerase chain reaction product for nitric oxide synthaserevealed a sequence that was identical to the cloned cDNA fromcytokine-activated rat aortic smooth muscle as described in Nunokawa,Y., et al, Biochem. Biophys. Res. Commun. 191, 89-94 (1993).

The results are shown in FIG. 2 wherein "LPS/IFN" stands for bacteriallipopolysaccharide/interferon-gamma; "AS" stands for argininosuccinatesynthetase; "AL" stands for argininosuccinate lyase; "NOS" stands fornitric oxide synthase; and "GAPDH" stands for glyceraldehyde-3-phosphatedehydrogenase.

FIG. 2 shows that argininosuccinate synthetase mRNA is not normallypresent at detectable levels in rat aortic smooth muscle cells butappears on treatment with immunostimulants when nitric oxide synthasemRNA appears. It further shows that argininosuccinate lyase mRNA isinitially present at detectable levels. Thus, the inducedargininosuccinate synthetase endows the cells with the capacity torecycle citrulline into arginine.

EXAMPLE III

Rat aortic smooth muscle cells were grown as described in Example I andwere untreated or treated with 30 μg/ml bacterial lipopolysaccharide incombination with 50 ng/ml interferon-gamma alone and with theconcentrations of mithramycin indicated in FIG. 3. After 6 hours, cellswere harvested and RNA was prepared and assayed by reversetranscriptase-polymerase chain reaction as described in Example II.Results using primers specific for glyceraldehyde-3-phosphatedehydrogenase as described in Example II and other primers specific forGTP cyclohydrolase I as described in Hattori, Y., et al, Biochem.Biophys. Res. Comm. 195, 435-441 (1993) are shown for comparison.

The results are depicted in FIG. 3 wherein "LPS/IFN" stands forbacterial lipopolysaccharide/interferon gamma; "AS" stands forargininosuccinate synthetase; "AL" stands for argininosuccinate lyase,"NOS" stands for nitric oxide synthase; "GTPCHi" stands for GTPcyclohydrolase I; and "GAPDH" stands for glyceraldehyde-3-phosphatedehydrogenase.

FIG. 3 shows that mithramycin inhibits gene transcription ofargininosuccinate synthetase and nitric oxide synthase but not of GTPcyclohydralase I, i.e., that mithramycin does not block all genetranscription but selectively inhibits transcription ofargininosuccinate synthetase and nitric oxide synthase.

EXAMPLE IV

Rat aortic smooth muscle cells were grown as described in Example I andwere continuously treated with 30 μg/ml of bacterial lipopolysaccharidein combination with 50 ng/ml of interferongamma. Concentrations ofmithramycin as indicated in FIG. 4 were added either simultaneously withimmunostimulant (filled in circles in FIG. 4) or 4 hours later (opencircles in FIG. 4).

Nitrite was used as an indicator of cellular NO synthesis and assay wascarried out as described in Gross, S. S., et al, J. Biol. Chem. 267,25722-25729 (1992). In the assay, cells were treated with serum- andarginine-free RPMI 1640 medium containing the desired test agents for 24hours. The accumulation of nitrite in the cell culture medium wasquantified colorimetrically after adding 100 μl of Griess reagent (1%sulfanilamide and 0.1% naphthalene diamine in 5% o-phosphoric acid) toan equal volume of sample.

The results are shown in FIG. 4 where points are mean values ±standarderror of mean (n=4) for nitrite accumulation in the cell culture mediumafter 24 hours. The results show that mithramycin inhibits induction ofNO synthetic activity but is not a direct inhibitor of argininosuccinatesynthetase or nitric oxide synthase. This is indicated because theresults show that if induction is allowed to proceed for 4 hours so thatargininosuccinate synthetase and nitric oxide synthase are present,mithramycin effectiveness to block NO production is markedly diminished.

Similar results indicating blocking of induction but failure to inhibitenzyme activities is shown when chromomycins in the same concentrationsor olivomycins in concentrations up to five times higher replacemithramycin.

EXAMPLE V

Rat aortic smooth muscle cells were grown as described in Example I andwere treated with 30 μg/ml of bacterial lipopolysaccharide incombination with 50 ng/ml of interferon-gamma. Concentrations ofL-citrulline and L-arginine as indicated in FIG. 5 were addedsimultaneously with immunostimulant. Nitrite was used as an indicator ofcellular NO synthesis and assay therefor was carried out as described inExample IV.

The results are shown in FIG. 5 where points are mean values ±standarderror of mean (n=4) of nitrite concentration measured in the cellculture medium after 24 hours.

The results show that, due to induction of argininosuccinate synthetaseactivity, cells can utilize L-citrulline just as efficiently asL-arginine to provide a substrate for nitric oxide production. Whenthere is 0 or low L-arginine, maximal nitrite is obtained (i.e., thesame nitrite production as with unlimited arginine) if the concentrationof L-citrulline is sufficiently high. Thus blocking L-citrullinesynthesis diminishes cells' capacity to make nitric oxide sinceL-arginine is being consumed by nitric oxide synthase.

Cells normally obtain L-arginine from diet or via circulation fromsynthesis in the kidney. While some L-arginine is supplied by thekidney, it is not the maximally usable amount. The experiment shows thatcells can make L-arginine from L-citrulline to supply the maximallyusable amount.

EXAMPLE VI

Immunostimulant treated rat aortic smooth muscle cells prepared as inExample I were homogenized in 10 volumes (10 ml/gm rat aortic smoothmuscle cells) of 100 mM tris buffer containing 1 mM phenylmethylsulfonylfluoride (a protein synthesis inhibitor) and 1 mM dithiothreitol (tomaintain enzyme in reduced form).

For the control, the crude homogenate was assayed for argininosuccinatesynthetase by the procedure as described in Example I except thatL-citrulline was present at a concentration of 150 μM instead of 5 mM.In duplicate runs, counts per minute were 534.00 (8.65% error) and443.00 (9.50% error) counts per minute.

When L-thiocitrulline was included at a concentration of 1 mM, countsper minute in the argininosuccinate synthetase assay in duplicate runswere 272.00 (12.13% error) and 227.00 (13.27% error) counts per minute.

When L-canavanine was included at a concentration of 1 mM, counts perminute in the argininosuccinate synthetase assay in duplicate runs were355.00 (10.61% error) and 393.00 (10.09% error) counts per minute.

When N.sup.ω -methyl-L-arginine was included at a concentration of 1 mM,counts per minute in the argininosuccinate synthetase assay in duplicateruns were 591.00 (8.23% error) and 520.00 (8.77% error) counts perminute.

The background count to be subtracted from the above count results wasobtained by omitting citrulline from the argininosuccinate synthetaseassay and in three runs was determined to be 217, 237 and 252 counts perminute.

The experiment shows that L-thiocitrulline and L-canavanine areinhibitors of immunostimulant induced argininosuccinate synthetase inrat aortic smooth muscle cells but that N^(G) -methyl-L⁻ arginine isnot.

When N.sup.ω -methyl-L-citrulline at a concentration of 1 mM isincluded, inhibition of immunostimulant induced argininosuccinatesynthetase is shown which is more potent than obtained with L-canavaninebut less potent than obtained with L-thiocitrulline is obtained.

When L-norvaline at a concentration of 1 mM is included, inhibition ofimmunostimulant induced argininosuccinate synthetase is shown.

When D-aspartate at a concentration of 1 mM is included, inhibition ofimmunostimulant induced argininosuccinate synthetase is shown.

EXAMPLE VII

A patient with Gram negative septicemia and blood pressure of 80/55 mmHg is administered mithramycin at a dose of 25 μg/kg intravenously in 50ml of saline over a 3 to 6 hour interval. The blood pressure returns tonormal range within 12 to 48 hours.

Similar results are obtained when chromamycin antibiotic complex at adose of 25 μg/kg or olivomycin antibiotic complex at a dose of 50 μg/kgis substituted for the mithramycin.

EXAMPLE VIII

A human is treated for renal cell cancer with interleukin-2 (1×10⁶units) for 5 days. By the fifth day, the patient's blood pressure fallsto 80/55 mm Hg and the patient encounters nausea and extreme discomfort.Three weeks later, the patient returns for a second course ofinterleukin-2 treatment but is pretreated (before commencinginterleukin-2 infusion) with mithramycin (25 μg/kg) and is treated at 24hour intervals thereafter with 25 μg/kg of mithramycin. The mithramycinis administered intravenously in 50 ml saline over a 3 to 6 hourinterval. When mithramycin is administered, hypotension does notdevelop.

Similar results to what are obtained with mithramycin are obtained whenchromomycin antibiotic complex at a dose of 25 μg/kg or olivomycinantibiotic complex at a dose of 50 μg/kg is substituted for themithramycin.

EXAMPLE IX

A patient with Gram negative septicemia and blood pressure of 80/55 mmHg is administered dopamine at 10 μg/kg/min to acutely increase bloodpressure and concurrently is administered mithramycin (25 μg/kg)intravenously in 50 ml saline over 3-6 hours. Blood pressure increasesacutely to the mild hypotensive range and the patient becomesnormotensive by 12 to 48 hours.

Similar results to what are obtained with mithramycin are obtained whenchromomycin antibiotic complex at a dose of 25 μg/kg or olivomycinantibiotic complex at a dose of 50 μg/kg is substituted for themithramycin.

EXAMPLE X

A patient with Gram negative septicemia and blood pressure of 80/55 mmHg is administered N^(G) -methyl-L-arginine in a bolus dose of 20 mg/kgintravenously in saline and thereafter is given a sustained infusion ofN^(G) -methyl-L-arginine at a dosage of 10 mg/kg/hr. Mithramycin (25μg/kg) is given intravenously in 50 ml saline over 3-6 hours starting atthe time of the bolus dose of N^(G) -methyl-L-arginine. Blood pressurebecomes normotensive within minutes of administration of the bolus doseof N^(G) -methyl-L-arginine and the normotensive state is sustained. Thepatient is released after 2 days.

Similar results to what are obtained with mithramycin are obtained whenchromomycin antibiotic complex at a dose of 25 μg/kg or olivomycinantibiotic complex at a dose of 50 μg/kg is substituted for themithramycin.

EXAMPLE XI

A patient with rheumatoid arthritis with swelling localized to twopainful joints is administered mithramycin at a dosage of 0.25 μg/kgintra-articularly into each joint. Local synovitis is reduced in 12 to24 hours.

Similar results to what are obtained with mithramycin are obtained whenchromomycin antibiotic complex at a dose of 0.25 μg/kg or olivomycinantibiotic complex at a dose of 0.50 μg/kg is substituted for themithramycin.

EXAMPLE XII

Sprague-Dawley rats are injected with 25 cc air subdermally in thedorsal area in accordance with the air pouch inflammatory model (Selye,H., Proc. Soc. Exper. Biol. and Med., 82, 328-333 (1953). Into the airpouch formed, an inflammatory stimulus, croton oil (0.5% in 0.5 ml cornoil), is injected. The rats in one group do not receive drug. The ratsin the second group receive mithramycin at 25 μg/kg intraperitoneallywith repeat doses at 24 hour intervals. At the end of 5 days, the groupof rats given mithramycin have significantly less nitrite in the fluidexudate contained in the granulomycous lesion and less inflammation thanthe rats in the other group.

Similar results to what are obtained with mithramycin are obtained whenchromomycin antibiotic complex at a dose of 25 μg/kg or olivomycinantibiotic complex at a dose of 50 μg/kg is substituted for themithramycin.

EXAMPLE XIII

A patient with Gram negative septicemia and blood pressure of 80/55 mmHg is administered L-thiocitrulline in a bolus dose of 20 mg/kgintravenously in saline and thereafter is given a sustained infusion ofL-thiocitrulline at a dose of 10 mg/kg/hr. Blood pressure becomesnormotensive within 5 to 10 minutes of administration of the bolus doseof L-thiocitrulline and the normotensive state is sustained. The patientis released after 2 days.

Similar results are obtained to what are obtained with L-thiocitrulline,when L-homothiocitrulline is used at double the dose forL-thiocitrulline. When N^(G) -methyl-L-citrulline or L-norvaline orL-isoleucine is used at quadruple the dose for L-thiocitrulline orD-aspartate is used at double the dose for L-thiocitrulline, bloodpressure is raised within about 1 to 4 hours of administration of thebolus dose.

EXAMPLE XIV

A human is treated for renal cell cancer with interleukin-2 (1×10⁶) for5 days. Simultaneously with commencing interleukin-2 infusion,L-thiocitrulline (10 mg/kg/day) is administered. Hypotension does notdevelop.

Similar results to what are obtained with L-thiocitrulline are obtainedwith the double dose for L-thiocitrulline of D-aspartate or quadruplethe dose for L-thiocitrulline of N.sup.ω -methyl-L-citrulline or N.sup.ω-ethyl-L-citrulline or L-norvaline or L-isoleucine.

EXAMPLE XV

A patient with Gram negative septicemia and blood pressure of 80/55 mmHg is administered dopamine at 10 μg/kg/min to acutely increase bloodpressure and concurrently is intravenously administered an initial bolusdose of L-thiocitrulline of 20 mg/kg and thereafter L-thiocitrulline asa sustained infusion at a dose of 10 mg/kg/hr. Blood pressure becomesnormal within minutes and this state is sustained. The patient isreleased within 2 days.

When the double the dose for L-thiocitrulline of D-aspartate orquadruple the dose for L-thiocitrulline of N.sup.ω -methyl-L-citrullineor L-norvaline or L-isoleucine are substituted for the L-thiocitrulline,blood pressure increases acutely to the mild hypotensive range and thepatient becomes normotensive by 24 to 72 hours.

EXAMPLE XVI

A patient with a Gram negative septicemia and blood pressure of 80/55 mmHg is administered N^(G) -methyl-L-arginine in a bolus dose of 20 mg/kgintravenously in saline and thereafter is given a sustained infusion ofN^(G) -methyl-L-arginine at a dose of 10 mg/kg/hr. N.sup.ω-Methyl-L-citrulline (40 mg/kg) is given intravenously in 50 ml salineover 3-6 hours starting at the time of the bolus dose of N^(G)-methyl-L-arginine. Blood pressure becomes mildly hypotensive or normalwithin minutes of administration of the bolus dose of N^(G)-methyl-L-arginine and this state is sustained.

Similar results are obtained as are obtained with N.sup.ω-methyl-L-citrulline when N.sup.ω -ethyl-L-citrulline at 40 mg/kg orD-aspartate at 20 mg/kg or L-norvaline at 20 mg/kg or L-isoleucine at 40mg/kg is given in place of the N.sup.ω -methyl-L-citrulline.

EXAMPLE XVII

A patient with rheumatoid arthritis with swelling localized to twopainful joints is administered L-thiocitrulline at a dose of 0.1 mg/kgintra-articularly into each joint. Local synovitis is reduced within 12to 72 hours.

When N.sup.ω -methyl-L-citrulline or L-norvaline or L-isoleucine isgiven intra-articularly into each joint at a dose of 0.2 mg/kg orD-aspartate is given at a dose of 0.1 mg/kg intra-articularly into eachjoint, local synovitis is reduced within 12 to 72 hours.

EXAMPLE XVIII

Sprague-Dawley rats are injected with 25 cc air subdermally in thedorsal area in accordance with the air pouch inflammatory model (Selye,H., Proc. Soc.. Exper. Biol. and Med., 82,328-333 (1953). Into the airpouch formed, an inflammatory simulus, croton oil (0.5% in 0.5 ml cornoil), is injected. The rats in one group do not receive drug. The ratsin the second group receive L-thiocitrulline at 20 mg/kgintraperitoneally with repeat doses at 6-24 hour intervals. At the endof 5 days, the group of rats given L-thiocitrulline have significantlyless nitrite in the fluid exudate contained in the granulomycous lesionand less inflammation than the rats in the other group.

Similar results as to what are obtained with L-thiocitrulline areobtained when 20 mg/kg of D-aspartate or 40 mg/kg of N.sup.ω-methyl-L-citrulline or N.sup.ω -propyl-L-citrulline or L-norvaline orL-isoleucine is substituted for the L-thiocitrulline.

EXAMPLE XIX

16.86 gm (0.1 mol) of L-ornithine hydrochloride is dissolved in 200 mlof 1 M NaOH. To the clear solution is added 5.71 gm of methyl isocyanate(0.1 mol) dropwise at room temperature. The pH is maintained at 9.5-10.5by occasional cautious additon of 1M NaOH. After stirring overnight atroom temperature, the reaction mixture is reduced to dryness by rotaryevaporation at reduced pressure. The residue is suspended inconcentrated HCl, and the precipitate of NaCl is filtered off. Thefiltrate, which contains N.sup.ω -methyl-L-citrulline is evaporated todryness under reduced pressure and redissolved in a small volume ofwater. That solution is applied to a column 2.0 by 50 cm of Dowex 50(Na⁺ form). The column is washed with 2 L of distilled water andfractions of approximately 25 ml are collected sequentially. Under theseconditions of chromatography, N.sup.ω -methyl-L-citrulline is eluted andresidual unreacted L-ornithine remains bound to the Dowex resin.Fractions containing N.sup.ω -methyl-L-citrulline (detected using aconventional ninhydrin spot assay) are pooled, rotary evaporated todryness under reduced pressure, and the residual oil is crystallizedfrom ethanol/water. The overall yield is approximately 75% based on theamount of ornithine used.

N.sup.ω -Alkyl-L-citrullines with different N.sup.ω -alkyl groups frommethyl are prepared by substituting an equivalent molar amount of thecorresponding alkyl isocyanate for the methyl isocyanate.

Many variations will be obvious to those skilled in the art. Thus, theinvention is defined by the claims.

What is claimed is:
 1. A method of prophylaxis or treatment of a subject for systemic hypotension caused by pathological overproduction of nitric oxide from arginine in vascular cells in said subject induced by therapy with a cytokine or by exposure to a bacterial endotoxin, said method comprising administering to a subject expected to develop or having such systemic hypotension a therapeutically effective amount of an argininosuccinate synthetase activity reducing agent.
 2. The method of claim 1 wherein the argininosuccinate synthetase activity reducing agent is an argininosuccinate synthetase induction blocking agent.
 3. The method of claim 2 wherein the argininosuccinate synthetase induction blocking agent is also a nitric oxide synthase induction blocking agent and is an antibiotic that binds to DNA sequences present in the upstream regulatory region of the argininosuccinate synthetase gene.
 4. The method of claim 3 wherein the antibiotic is selected from the group consisting of mithramycin, chromomycins and olivomycins.
 5. The method of claim 4 wherein the antibiotic is mithramycin.
 6. The method of claim 1 wherein the argininosuccinate synthetase activity reducing agent is an argininosuccinate synthetase inhibitor.
 7. The method of claim 6 wherein the argininosuccinate synthetase inhibitor is an L-citrulline antagonist.
 8. The method of claim 7 wherein the L-citrulline antagonist is not L-thiocitrulline or L-homothiocitrulline.
 9. The method of claim 8 wherein the L-citrulline antagonist is N.sup.ω -alkyl-L-citrulline wherein the alkyl contains 1 to 6 carbon atoms.
 10. The method of claim 9 wherein the L-citrulline antagonist is N.sup.ω -methyl-L-citrulline.
 11. The method of claim 6 wherein the argininosuccinate synthetase inhibitor is an L-aspartate antagonist.
 12. The method of claim 11 wherein the L-aspartate antagonist is D-aspartate.
 13. A method for treatment of a subject for systemic hypotension caused by pathological overproduction of nitric oxide from arginine in vascular cells in said subject induced by therapy with a cytokine or by exposure to a bacterial endotoxin, said method comprising administering to a subject having such systemic hypotension a therapeutically effective amount of an α₁ -adrenergic agonist and an amount of argininosuccinate synthetase activity reducing agent to restore vascular contractile sensitivity to the effects of the α₁ -adrenergic agonist.
 14. The method of claim 13 wherein the argininosuccinate synthetase activity reducing agent is an argininosuccinate synthetase induction blocking agent.
 15. The method of claim 14 wherein the argininosuccinate synthetase induction blocking agent is also a nitric oxide synthase induction blocking agent and is an antibiotic that binds to DNA sequences present in the upstream regulatory region of the argininosuccinate synthetase gene.
 16. The method of claim 15 wherein the antibiotic is selected from the group consisting of mithramycin, chromomycins and olivomycins.
 17. The method of claim 16 wherein the antibiotic is mithramycin.
 18. The method of claim 13 wherein the argininosuccinate synthetase activity reducing agent is an argininosuccinate synthetase inhibitor.
 19. The method of claim 18 wherein the argininosuccinate synthetase inhibitor is an L-citrulline antagonist.
 20. The method of claim 19 wherein the L-citrulline antagonist is not L-thiocitrulline or L-homothiocitrulline.
 21. The method of claim 20 wherein the L-citrulline antagonist is N.sup.ω -alkyl-L-citrulline wherein the alkyl contains 1 to 6 carbon atoms.
 22. The method of claim 21 wherein the L-citrulline antagonist is N.sup.ω -methyl-L-citrulline.
 23. The method of claim 18 wherein the argininosuccinate synthetase inhibitor is an L-aspartate antagonist.
 24. The method of claim 23 wherein the L-aspartate antagonist is D-aspartate.
 25. A method for treating a subject for systemic hypotension caused by pathological overproduction of nitric oxide from arginine in vascular cells in said subject induced by therapy with a cytokine or by exposure to a bacterial endotoxin, said method comprising administering to said subject a therapeutically effective amount of an arginine antagonist of nitric oxide synthesis by nitric oxide synthase and a therapeutically effective amount of an argininosuccinate synthetase activity reducing agent selected from the group consisting of orgininosuccinate synthetase induction blocking agents and argininosuccinate synthetase inhibitors which do not block the activity of inducible nitric oxide synthase.
 26. The method of claim 25 wherein the argininosuccinate synthetase activity reducing agent is an arginiosuccinate synthetase induction blocking agent.
 27. The method of claim 26 wherein the argininosuccinate synthetase induction blocking agent is also a nitric oxide synthase induction blocking agent and is an antibiotic that binds to DNA sequences present in the upstream regulatory region of the argininosuccinate synthetase gene.
 28. The method of claim 27 wherein the antibiotic is selected from the group consisting of mithramycin, chromomycins and olivomycins.
 29. The method of claim 28 wherein the antibiotic is mithramycin.
 30. The method of claim 25 wherein the argininosuccinate synthetase activity reducing agent is an argininosuccinate synthetase inhibitor which does not block the activity of inducible nitric oxide synthase.
 31. The method of claim 30 wherein the argininosuccinate synthetase activity reducing agent is an L-citrulline antagonist.
 32. The method of claim 31 wherein the L-citrulline anagonist is N.sup.ω -alkyl-L-citrulline wherein the alkyl contains 1 to 6 carbon atoms.
 33. The method of claim 32 wherein the L-citrulline antagonist is N.sup.ω -methyl-L-citrulline.
 34. The method of claim 30 wherein the argininosuccinate synthetase activity reducing agent is an L-aspartate antagonist.
 35. The method of claim 34 wherein the L-aspartate antagonist is D-aspartate. 